Gm14207/Dll4/Notch信号通路在造血干细胞移植骨髓血管龛重建中的调控作用研究

Prolonged low hemogram after hematopoietic stem cell transplantation (HSCT) which is dependent on recipients’ hematopoietic reconstitution seriously affects their quality of life. Damaged bone marrow sinusoidal endothelial cells during HSCT would directly influence the function of vascular niche, whose recovery is critical for hematopoietic reconstitution as bone marrow vascular niche regulates hematopoiesis. Dll4/Notch signaling is reported to regulate the regeneration of effective blood vessels and the mechanism by how this signaling works regarding transcriptional regulation remains unclear. Long non-coding RNA (lncRNA) has recently been identified as the new player in regulation of gene expression. Our previous study found that lncRNA Gm14207 has a higher homology to Dll4,a Notch ligand and their expression in bone marrow sinusoidal endothelial cells were decreased simultaneously and also positively correlated, suggesting Gm12407 is a positive regulator of Dll4. In this project, on the basis of understanding of the regulation of Notch1 expression by 0610009E02Rik, we propose to establish ex and in vivo model of endothelial cell damage to further investigate the role of Gm12407 in the regeneration of bone marrow blood vessels as well as the mechanism by how it regulates Dll4 expression from the aspect of epigenetic and protein translation, to broaden the knowledge on Notch1 signaling pathway. Deep analysis of the mechanism of bone marrow vascular niche reconstitution will provide experimental evidence for clinical studies on the prolonged low hemogram in recipients after HSCT.

造血干细胞移植后的长期低血象状态严重影响受者生存质量，此状态直接取决于受者造血重建。骨髓血管龛是造血的场所，移植中骨髓窦内皮细胞受损直接影响血管龛功能，其恢复是造血重建的关键，Dll4/Notch信号通路调控有效的血管再生，然而其转录调控的探索匮乏。长链非编码RNA是基因表达调控的新着眼点，我们发现长链非编码RNA Gm14207与Notch配体Dll4高度同源；移植中骨髓窦内皮细胞内Gm14207与Dll4同步下降，二者呈正相关，提示Gm14207是Dll4的正性调节子。故本课题拟在明确0610009E02Rik调控Notch1表达的基础上，建立内皮细胞损伤离体和在体模型，进一步探索Gm14207在骨髓血管再生中的作用，从表观遗传及蛋白翻译等方面探索其调控Dll4的表达机制，拓宽人们对Notch信号通路的认识，深入解析骨髓血管龛重建的机理，为移植受者长期低血象的临床研究提供实验依据。

造血干细胞移植后的长期低血象状态严重影响受者生存质量，此状态直接取决于受者造血重建。骨髓血管龛是造血的场所，移植中骨髓窦内皮细胞受损直接影响血管龛功能，其恢复是造血重建的关键，Dll4/Notch信号通路调控有效的血管再生，然而其转录调控的机制尚未明确。本实验主要研究微血管内皮细胞内长链非编码Gm14207 对Notch配体Dll4表达的调控机理，进一步明确 Gm14207对HSCT中骨髓血管niche重建的调控作用。根据内皮细胞增殖周期及白消安损伤内皮细胞的药物浓度及作用时间的确定，成功建立内皮细胞损伤模型。研究发现，lncRNA-Gm14207可促进损伤内皮细胞凋亡、抑制细胞增殖以及抑制其血管生成能力。白消安损伤过表达Gm4207内皮细胞后，Dll4 mRNA的表达量降低，Notch1及其lncRNA-RikmRNA的表达量亦降低；白消安损伤过表达Gm4207内皮细胞后抑制内皮细胞Notch1信号通路的下游靶基因Hes1的表达。上述结果提示lncRNA-Gm14207与Notch1/DLL4信号通路相互作用，参与了损伤内皮细胞的修复。研究发现还照射可促进内皮细胞发生间质化,内皮细胞间的通透性增高；损伤的内皮细胞抑制造血干细胞增殖、促进造血干细胞分化，说明损伤内皮细胞间质化和通透性的改变参与了造血干细胞的功能调控，提示内皮细胞间质化和通透性的有效干预，是促进造血重建的重要策略。此外，我们进一步研究发现NLRP1抑制骨髓造血干细胞增殖和分化；NLRP1抑制骨髓血管龛的生长，提示NLRP1参与了骨髓血管龛对造血重建的调控。但具体的机制有待深入探索。本实验拓展了人们对骨髓血管龛的有效血管再生、重建的认识，分析了骨髓血管龛损伤修复重建的机理，为临床移植后造血延迟的治疗提供理论与实验依据。